Post-translational processing of bioactive peptides is an area of significant importance and of growing interest. In this study, we hypothesize that the proenkephalin A (Proenk A) family of peptides undergo in vivo phosphorylation, sulfation, and acetylation and that these chemical modifications are essential in maintaining homeostasis in living organisms. Furthermore, the lesions in these peptidergic pathways result from or lead to pathophysiological disorders and the degree of the chemical modification can be correlated to a pathophysiology. Neuropeptides are synthesized in the cell body initially as large precursor proteins, which undergo proteolytic cleavage and post-translational modifications to produce mature peptides. Knowledge of this entire metabolic sequence is of practical importance. To test our hypothesis, the experiments are planned to synthesize those modified neuropeptides, to develop structure-specific and sensitive mass spectrometry (MS) techniques, and to identify structurally those modified neuropeptides in biological (human, bovine, and porcine) fluids and tissues. Defining the molecular form of a neuropeptide, and knowing its concentration in a given tissue is important to understand the functional role of that peptide in that tissue. The specific aims of this research proposal are: (a)to synthesize phosphorylated, sulfated, and acetylated Proenk A peptides. These modified peptides are required as model reference compounds to study their MS properties and to develop extraction/purification procedures, and as substrates to study their enzymatic degradation, (b)to develop highly structure-specific and sensitive analytical techniques employing liquid secondary ion MS (LSIMS) and electrospray (ES) MS and MS/MS methods. This objective will provide us means to obtain an accurate knowledge of the primary structure of the modified Proenk A peptides, (c)to analyze those modified neuropeptides in the extracts of biological fluids and tissues, and (d)to study the enzymatic degradation of those modified neuropeptides by utilizing CSF or plasma as the source of endopeptidases. This objective will shed light on the possible role of those chemical modifications in enhancing the proteolytic stability. The use of structure-specific MS methods to analyze human fluids and tissues is a right step in our understanding of human health. At present, completely satisfactory structure-specific methods for phosphorylated and sulfated peptides are lacking, and this study will fill that gap.